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Array-Based Online Two Dimensional Liquid Chromatography System Applied to Effective Depletion of High-Abundance Proteins in Human Plasma

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Download Hi-Res ImageDownload to MS-PowerPointCite This:Anal. Chem. 2016, 88, 4, 2440-2445
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    Array-Based Online Two Dimensional Liquid Chromatography System Applied to Effective Depletion of High-Abundance Proteins in Human Plasma
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    † Department of Chemistry, Fudan University, Shanghai 200433, China
    ‡ Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China
    *Tel: +86-21-55664165. Fax: 86-21-65641740. E-mail: [email protected] (X. Zhang)
    *Tel: +86-21-65643983. Fax: +86-21-65641740. E-mail: [email protected] (M. Gao)
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    Analytical Chemistry

    Cite this: Anal. Chem. 2016, 88, 4, 2440–2445
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    https://doi.org/10.1021/acs.analchem.5b04553
    Published January 19, 2016
    Copyright © 2016 American Chemical Society
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    Abstract

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    In this work, an array-based online two-dimensional liquid chromatography (2D-LC) system was constructed for protein separation and effective depletion of high-abundance proteins in human plasma. This system employed a strong anion exchange column in the first dimension and eight reversed-phase liquid chromatographic columns in the second dimension. All the protein components in the first dimension were enriched on the trapping columns, simultaneously back-flushed and concurrently separated in the second dimension. LC eluents were then collected on 96-well plates for further analysis. Compared with common 2D-LC system, this system showed an 8-fold increase in throughput and convenient utilization of stop-flow mode for sample separation. The RSD of retention time and peak area were separately below 0.51% and 8%. Recovery rates of four standard proteins were all above 95%. This array-based 2D-LC system was subsequently applied to the analysis of proteins in human plasma. The eluents containing high-abundance proteins were rapidly located according to the results of bicinchoninic acid assay. In all, with the effective depletion of 84 high-abundance proteins, a total of 1332 proteins were identified through our system. The dynamic range of the identified protein concentrations covered 9 orders of magnitude, ranging from 41 g/L level for HSA down to 0.01 ng/mL level for the low-abundance proteins.

    Copyright © 2016 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04553.

    • Details of experimental procedures and additional data of performance evaluation of this array-based 2D-LC system (PDF)

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    Analytical Chemistry

    Cite this: Anal. Chem. 2016, 88, 4, 2440–2445
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.analchem.5b04553
    Published January 19, 2016
    Copyright © 2016 American Chemical Society
    Request reuse permissions

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